Research reportAcute systemic administration of interleukin-10 suppresses the beneficial effects of moderate hypothermia following traumatic brain injury in rats
Introduction
Traumatic brain injury (TBI) produces long-term disturbances in neurobehavioral and cognitive function. One mechanism for the detrimental effects may be the initiation of inflammatory processes such as the synthesis of proinflammatory cytokines that are implicated in secondary tissue damage. Experimental and clinical studies of TBI have shown robust inflammatory responses, including the early production of cytokines [52] and the upregulation of (E)-selectin and intercellular adhesion molecule-1 (ICAM-1) on cerebrovascular endothelial cells [7], [41]. Previous studies using the controlled cortical impact (CCI) or fluid percussion (FP) injury models of TBI have shown that neutrophils accumulate in the brain as early as 4 h after injury and reach peak levels by 24–72 h [7], [8], [11], [47]. Injury severity, brain edema, and blood–brain barrier (BBB) disruption are some of the factors contributing to neutrophil accumulation [47].
Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that inhibits a variety of macrophage responses including the synthesis of cytokines, adhesion molecules, and chemokines. IL-10 mediates these effects through both signal transducers and activators of transcription (STAT)-3 and nuclear factor kappa-B (NF-κB)-mediated effects [42], [44]. IL-10 is also a potent suppressor of T-lymphocyte proliferation and cytokine responses via selected inhibition of CD28 tyrosine phosphorylation [1].
IL-10 has been studied in several central nervous system (CNS) injury models [3], [5], [17], [32], [45], [48]. Exogenously administered IL-10 has been shown to reduce tumor necrosis factor-alpha (TNF-α) production and lesion volume, and to significantly improve functional recovery following spinal cord injury in rats [3]. Neurological protection has also been reported in middle cerebral artery occluded rats administered IL-10 centrally into the lateral ventricle 30 min and 3 h after injury [48]. Beneficial effects of IL-10 administration were shown after experimental TBI in rats. Knoblach and Faden [32] reported that intravenous administration of IL-10 30 min before and 1 h after FP injury in rats improved neurobehavioral outcome at both 1 and 2 weeks [32].
The proinflammatory response to TBI may be attenuated by strategies other than the administration of anti-inflammatory cytokines. Hypothermia may also attenuate TBI-induced inflammatory responses. Whalen and colleagues [50] have shown that hypothermia initiated 10 min after CCI injury attenuated neutrophil influx into the injured brain. Chatzipanteli et al. [8] have reported similar results after FP injury. More recently, Gundersen et al. [23] reported that moderate hypothermia (32.5–33.0 °C) blunts the inflammatory response to acute hemorrhage in rats by reducing interleukin-6 (IL-6) levels and diminishing reactive oxygen species. In addition to the potentially beneficial effect of reducing injury-induced inflammation, hypothermia has already been proven to benefit outcome after TBI. Improved functional and/or histological outcomes have been demonstrated following FP [4], [14], [34], weight drop [33], and CCI [10], [19], [53] injury. In humans, significant reductions in intracranial pressure, as well as improved outcome have been shown with moderate hypothermia [12], [36]. Despite the negative finding from a multicenter trial examining the effects of hypothermia following TBI [13], there is still optimism that hypothermia is a viable approach to improving outcome after TBI in humans and is still used as a therapy to control refractory intracranial hypertension. The contribution of the suppression of inflammation by hypothermia remains to be determined after both experimental and clinical TBI. The combination of the administration of anti-inflammatory cytokines with hypothermia may prove to be a promising therapeutic strategy following TBI.
Despite considerable promise for combination therapies in experimental CNS injury [2], [35], [53], several studies indicate that combined therapies are not always effective [37], [46] and in some cases may actually be counter productive [20], [22]. The efficacy of IL-10 coupled with moderate hypothermia following TBI is unknown. However, in a rat ischemia model the anti-inflammatory agent dipyrone has been shown to improve long-term outcomes when added to hypothermia therapy [15]. Additionally, postischemic hypothermia and IL-10 treatment have been shown to provide long-lasting neuroprotection of hippocampal CA1 neurons following transient global ischemia in rats [17]. The aim of this study is to test the hypothesis that the combination of IL-10 and moderate hypothermia will provide synergistic neuroprotective effects after TBI.
Section snippets
Animals
Sixty-five adult male Sprague–Dawley rats (Harlan, Inc., Indianapolis, IN) were housed in standard steel-wire mesh cages and maintained in a temperature (21±1 °C) and light (on 7:00 to 19:00 h) controlled environment with food and water available ad libitum. All experimental procedures were approved by the Animal Care and Use Committee at the University of Pittsburgh and were conducted in accordance with the recommendations provided in the “Guide for the Care and Use of Laboratory Animals
Temperature regulation
All rats in the hypothermic conditions reached the target temperature of 32±0.3 °C within 15 min after CCI injury (range=10.25 to 14.68 min for TBI/VEH and 10.34–14.45 min for TBI/IL-10; Fig. 1). After 3 h of hypothermia the animals were warmed 0.8–0.9 °C every 10 min for 1 h until reaching the baseline normothermic temperature of 37±0.3 °C. There were no significant differences in time to reach the target temperatures between the hypothermic groups. The normothermic groups were maintained at
Discussion
The aim of the present study was to test the hypothesis that systemic administration of the anti-inflammatory cytokine IL-10 combined with moderate hypothermia (32 °C) would provide synergistic neuroprotective effects after TBI. Contrary to our hypothesis, the IL-10 plus hypothermia regimen did not improve outcome following TBI. Moreover, the administration of IL-10 under normothermic conditions was ineffective in protecting against motor and cognitive impairment after TBI as evidenced by a lack
Note in proof
In a preliminary report (J. Neurotrauma, 18:1175, 2001), Bramwell et al. reported on the effects of interleukin-10 (IL-10) on functional and histological outcome after fluid percussion (FP) brain injury in rats. A single intraperitoneal injection of IL-10 (5 mg) was given 30 min. after injury to rats maintained at normothermic (37 °C) or mildly hypothermic (34 °C) conditions for 4 hrs. Functional and histological outcome revealed that neither the IL-10 nor IL-10 plus hypothermia groups exhibited
Acknowledgements
The authors thank Dr Warner Lo, Ohio State University, for providing the hybridoma for the generation of RP-3. We also thank Michael J. Whalen, MD and Darcy Franicola, BS for assistance with the immunohistochemistry. This work was supported by NIH grants NS33150, NS30318, NS40125 (CED) and by the Walter L. Copeland Fund (# D 2000-0253) of The Pittsburgh Foundation (AEK).
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Dr Anthony E. Kline is currently in the Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USA.